The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggregation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute significantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both all-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.
protein aggregation; molecular dynamics; protein folding; neurodegeneration
Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors and other proteins synthesized as inactive precursors. Serine proteases are one of the largest and conserved families of endopeptidases involved in diverse cellular activities including wound healing, blood coagulation and immune responses. Heteromeric α,β,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle’s syndrome, are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/Channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that is previously unidentified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC, and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. Based on our results, we propose a dynamics-driven “anglerfish” mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase compared to furin.
ENaC; serine endopeptidase; Xenopus; voltage clamp; discrete molecular dynamics
Molecular modeling of proteins including homology modeling, structure determination, and knowledge-based protein design requires tools to evaluate and refine three-dimensional protein structures. Steric clash is one of the artifacts prevalent in low-resolution structures and homology models. Steric clashes arise due to the unnatural overlap of any two non-bonding atoms in a protein structure. Usually, removal of severe steric clashes in some structures is challenging since many existing refinement programs do not accept structures with severe steric clashes. Here, we present a quantitative approach of identifying steric clashes in proteins by defining clashes based on the Van der Waals repulsion energy of the clashing atoms. We also define a metric for quantitative estimation of the severity of clashes in proteins by performing statistical analysis of clashes in high-resolution protein structures. We describe a rapid, automated and robust protocol, Chiron, which efficiently resolves severe clashes in low-resolution structures and homology models with minimal perturbation in the protein backbone. Benchmark studies highlight the efficiency and robustness of Chiron compared to other widely used methods. We provide Chiron as an automated web server to evaluate and resolve clashes in protein structures that can be further used for more accurate protein design.
Homology modeling; refinement; Chiron; Discrete Molecular Dynamics; Protein Design
Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling.
The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multi-domain membrane protein which matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32 residue segment termed the regulatory insertion (RI) not present in other ABC transporters. We report here that RI deletion enabled ΔF508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14h in cells, similar to the wild-type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermo-stabilizing influence on the channel with gating up to at least 40°C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508 containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.
ABC transporters; CFTR; cystic fibrosis; ion channel; DMD simulations
The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTR’s disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF–causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1–ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.
Improved knowledge of the dynamics within CFTR—including the R region and NBD1—will expand our understanding of its function, as well as the implications of the F508del defect that is present in many cystic fibrosis patients.
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain incomplete. Here we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild-type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the SDR, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein’s inherent stability and channel activity returns a near-normal state.
ABC transporters; CFTR; protein thermal stability; ion channel; DMD simulations
Motivation: Increasing use of structural modeling for understanding structure–function relationships in proteins has led to the need to ensure that the protein models being used are of acceptable quality. Quality of a given protein structure can be assessed by comparing various intrinsic structural properties of the protein to those observed in high-resolution protein structures.
Results: In this study, we present tools to compare a given structure to high-resolution crystal structures. We assess packing by calculating the total void volume, the percentage of unsatisfied hydrogen bonds, the number of steric clashes and the scaling of the accessible surface area. We assess covalent geometry by determining bond lengths, angles, dihedrals and rotamers. The statistical parameters for the above measures, obtained from high-resolution crystal structures enable us to provide a quality-score that points to specific areas where a given protein structural model needs improvement.
Availability and Implementation: We provide these tools that appraise protein structures in the form of a web server Gaia (http://chiron.dokhlab.org). Gaia evaluates the packing and covalent geometry of a given protein structure and provides quantitative comparison of the given structure to high-resolution crystal structures.
Supplementary information: Supplementary data are available at Bioinformatics online.
We developed a new system for light-induced protein dimerization in living cells using a novel photocaged analog of rapamycin (pRap) together with an engineered rapamycin binding domain (iFKBP). Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.
Neurodegeneration, the progressive loss of function in neurons that eventually leads to their death, is the cause of many neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Protein aggregation is a hallmark of most neurodegenerative diseases, where unfolded proteins form intranuclear, cytosolic, and extracellular insoluble aggregates in neurons. Mounting evidence from studies in neurodegenerative disease models shows that molecular chaperones, key regulators of protein aggregation and degradation, play critical roles in the progression of neurodegeneration. Although chaperones exhibit promiscuity in their substrate specificity, specific molecular features are required for substrate recognition. Understanding the basis for substrate recognition by chaperones will aid in the development of therapeutic strategies that regulate chaperone expression levels in order to combat neurodegeneration. Many experimental techniques, including alanine scanning mutagenesis and phage display library screening, have been developed and applied to understand the basis of substrate recognition by chaperones. Here, we present computational algorithms that can be applied to rapidly screen the sequence space of potential substrates to determine the sequence and structural requirements for substrate recognition by chaperones.
The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.
Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity, but does not affect other protein interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases.
Mammalian airways are protected from infection by a thin film of airway surface liquid (ASL) which covers airway epithelial surfaces and acts as a lubricant to keep mucus from adhering to the epithelial surface. Precise regulation of ASL volume is essential for efficient mucus clearance and too great a reduction in ASL volume causes mucus dehydration and mucus stasis which contributes to chronic airway infection. The epithelial Na+ channel (ENaC) is the rate-limiting step that governs Na+ absorption in the airways. Recent in vitro and in vivo data have demonstrated that ENaC is a critical determinant of ASL volume and hence mucus clearance. ENaC must be cleaved by either intracellular furin-type proteases or extracellular serine proteases to be active and conduct Na+, and this process can be inhibited by protease inhibitors. ENaC can be regulated by multiple pathways, and once proteolytically cleaved ENaC may then be inhibited by intracellular second messengers such as cAMP and PIP2. In the airways, however, regulation of ENaC by proteases seems to be the predominant mode of regulation since knockdown of either endogenous serine proteases such as prostasin, or inhibitors of ENaC proteolysis such as SPLUNC1, has large effects on ENaC activity in airway epithelia. In this review, we shall discuss how ENaC is proteolytically cleaved, how this process can regulate ASL volume, and how its failure to operate correctly may contribute to chronic airway disease.
Na+ channel; Cystic fibrosis; Epithelial transport; Fluid absorption; Lung liquid